In depression, bacterial translocation may drive inflammatory responses, oxidative and nitrosative stress (O&NS), and autoimmune responses directed against O&NS-damaged neoepitopes.

OBJECTIVE: Depression is accompanied by activation of immuno-inflammatory and oxidative and nitrosative stress (IO&NS) pathways, and increased IgM/IgA responses to lipopolysaccharide (LPS) of gram-negative commensal bacteria. The latter suggests that bacterial translocation has caused IgM/IgA responses directed against LPS. Bacterial translocation may drive IO&NS responses. METHOD: To examine the associations between IgM/IgA responses to LPS and IO&NS measurements, including plasma/serum interleukin-1 (IL-1), tumor necrosis factor (TNF)α, neopterin, lysozyme, oxidized LDL (oxLDL) antibodies, peroxides, and IgM (auto)immune responses against malondialdehyde (MDA), azelaic acid, phophatidyl inositol (Pi), NO-tryptophan and NO-tyrosine in depressed patients and controls. RESULTS: We found significant positive associations between IgM/IgA responses to LPS and oxLDL antibodies, IgM responses against MDA, azelaic acid, Pi, NO-tryptophan, and NO-tyrosine. The IgA responses to LPS were correlated with lysozyme. There were no significant positive correlations between the IgM/IgA responses to LPS and IL-1 and neopterin. CONCLUSION: The findings show that in depression there is an association between increased bacterial translocation and lysozyme production, an antibacterial compound, O&NS processes, and autoimmune responses directed against O&NS generated neoantigenic determinants. It is suggested that bacterial translocation may drive IO&NS pathways in depression and thus play a role in its pathophysiology.

Direct visualization of retinoic acid in the rat hypothalamus: an immunohistochemical study.

In order to increase our knowledge about the distribution of vitamins in the mammalian brain, we have developed a highly specific antiserum directed against retinoic acid with good affinity (10(-8) M), as evaluated by ELISA tests. In the rat brain, no immunoreactive fibers containing retinoic acid were detected. Cell bodies containing retinoic acid were only found in the hypothalamus. This work reports the first visualization and the morphological characteristics of cell bodies containing retinoic acid in the mammalian paraventricular hypothalamic nucleus and in the dorsal perifornical region, using an indirect immunoperoxidase technique. The restricted distribution of retinoic acid in the rat brain suggests that this vitamin could be involved in very specific physiological mechanisms.

Immunocytochemical visualization of D-glutamate in the rat brain.

Using highly specific antisera directed against conjugated d-amino acids, the distribution of d-glutamate-, d-tryptophan-, d-cysteine-, d-tyrosine- and d-methionine-immunoreactive structures in the rat brain was studied. Cell bodies containing d-glutamate, but not d-glutamate-immunoreactive fibers, were found. Perikarya containing this d-amino acid were only found in the mesencephalon and thalamus of the rat CNS. Thus, the highest density of cell bodies containing d-glutamate was observed in the dorsal raphe nucleus, the ventral part of the mesencephalic central gray, the superior colliculus, above the posterior commissure, and in the subparafascicular thalamic nucleus. A moderate density of immunoreactive cell bodies was observed in the dorsal part of the mesencephalic central gray, above the rostral linear nucleus of the raphe, the nucleus of Darkschewitsch, and in the medial habenular nucleus, whereas a low density was found below the medial forebrain bundle and in the posterior thalamic nuclear group. Moreover, no immunoreactive fibers or cell bodies were visualized containing d-tryptophan, d-cysteine, d-tyrosine or d-methionine in the rat brain. The distribution of d-glutamate-immunoreactive cell bodies in the rat brain suggests that this d-amino acid could be involved in several physiological mechanisms. This work reports the first visualization and the morphological characteristics of conjugated d-glutamate-immunoreactive cell bodies in the rat CNS using an indirect immunoperoxidase technique. Our results suggest that the immunoreactive neurons observed have an uptake mechanism for d-glutamate.

Circulating antibodies to cysteinyl catecholamines in amyotrophic lateral sclerosis and Parkinson's disease patients.

Amyotrophic lateral sclerosis (ALS) is a degenerative disease of unknown aetiology, affecting motor neurons. Many radical species, such as O(2)(-) NO, and ONOO(-), and lipoperoxidative products are involved, but not all processes have yet been identified. It is known that the oxidation of catecholamines leads to quinone formation. These orthoquinones react with the sulphhydril group of cysteine to produce neurotoxic cysteinyl catecholamine (Cyst-CA) neo-compounds. We synthesised Cyst-CA in order to mimic their endogenous formation. Using the ELISA method, circulating antibodies to Cyst-CA were found in sporadic ALS sera. First, the antibody titres were compared to those of controls and patients with other neurodegenerative diseases. Significant antibody levels were found for Cyst-CA. The G and A isotypes were found but not the M isotype. A second series of experiments showed that A and G titres were elevated, depending on the type of Cyst-CA and the onset of the disease. IgG to Cyst-3,4-dihydroxyphenylalanine (L-DOPA) were present in cases of bulbar and upper limb onsets. IgA to Cyst-homovanillic acid (HVA), Cyst-adrenaline (A), and Cyst-dopamine (DA) were found in lower limb onset. These results indirectly show that: 1) the oxidation of CA and the formation of Cyst-CA may be involved in ALS; 2) these radical processes have different targets depending on the onset of the disease.

Antibodies directed against nitrosylated neoepitopes in sera of patients with human African trypanosomiasis.

Antibodies directed against nitrosylated epitopes have been found in sera from patients suffering from human African trypanosomiasis (HAT) but not in sera from control subjects living in the same endemic area or African control subjects living in France. We conjugated amino acids to albumin by glutaraldehyde (conjugates) and then nitrosylated the conjugates. Both conjugates and nitrosylated conjugates were analysed by enzyme-linked immunosorbent assay (ELISA). We detected antibodies directed against nitrosylated L-cysteine and L-tyrosine conjugates; antibody levels were higher in stage II patients than in stage I. Patients with severe clinical signs had higher antibody levels, and antibody levels were highest in patients with major neurological signs. Antibody response was only associated with the IgM isotype. We evaluated antibody specificity and avidity by competition experiments using conjugates and nitrosylated conjugates. Avidity was around 2 x10(-6) m for the S-nitroso-cysteine epitope and 2 x 10(-8) m for the S-nitroso-tyrosine epitope. Detection of circulating antibodies to S-nitroso-cysteine and S-nitroso-tyrosine epitopes provides indirect evidence for nitric oxide (NO) involvement in HAT and their levels are correlated with disease severity.

Immunohistochemical localization of protein 3-nitrotyrosine and S-nitrosocysteine in a murine model of inhaled nitric oxide therapy.

Inhaled nitric oxide (INO) therapy is currently used clinically to selectively dilate the pulmonary vasculature and to help treat persistent pulmonary hypertension and bronchopulmonary dysplasia in the neonate. However, in the presence of oxygen or superoxide, nitric oxide forms potentially harmful reactive nitrogen species. Using an experimental mice model, we examined the effects of concurrent hyperoxia and INO on protein tyrosine nitration and cysteine S-nitrosylation in pulmonary tissue. Data showed enhanced 3-nitrotyrosine staining within the airway epithelium and alveolar interstitium of mice lungs treated with hyperoxia, which did not increase significantly with INO administration. Within the alveolar interstitium, 3-nitrotyrosine staining was localized to macrophages. S-Nitrosocysteine staining in airway epithelium was significantly enhanced with INO administration regardless of oxygen content. These data suggest that the formation of protein S-nitrosocysteine is the major protein modification during administration of INO.

Detection of nitrosylated epitopes in Trypanosoma brucei gambiense by polyclonal and monoclonal anti-conjugated-NO-cysteine antibodies.

Activated macrophages with the Calmette/Guérin bacillus (BCG) have a cytotoxic/cytostatic effect on the extracellular parasite, Trypanosoma brucei gambiense. This effect was inhibited when the NO-synthase inhibitor NG-monomethyl-L-arginine (NMMA; 0.5 mM) was added to the culture media. Using an immunocytochemical method with rabbit polyclonal or mouse monoclonal antibodies directed against conjugated nitroso-epitopes (anti-conjugated-NO-cysteine), nitrosylated antigens were visualized in fixed trypanosomes. These results suggest that NO was synthesized by the activated macrophages and that it reacted with some parasitic proteins containing cysteine. The release of NO bound to parasitic proteins may cause the killing of trypanosomes. The immunoreactivity was positive when the trypanosomes were obtained from the supernatant of the BCG-activated macrophages that contains BSA (4 mg/mL). In contrast, the parasites cocultured with non-activated macrophages remained completely viable, and, the immunoreactivity was completely negative.

Albumin nitrosylated by activated macrophages possesses antiparasitic effects neutralized by anti-NO-acetylated-cysteine antibodies.

Activated macrophages exert an L-arginine-dependent cytostatic effect on the extracellular parasite, Trypanosoma musculi. This effect is not observed in the absence of albumin in the culture medium but is restored by the addition of albumin, indicating the presence of an albumin-nitric oxide (NO) adduct acting as an effector molecule. Since L-cysteine represents a privileged target for NO, an immunochemical approach was performed using an acetylated-cysteine-BSA conjugate. This conjugate was nitrosylated using sodium nitrite as a NO donor. Binding of NO to the conjugated haptens was assayed using spectrophotometry. It was completely abolished by mercuric chloride, confirming the presence of an S-NO bond. Polyclonal Abs were obtained after immunizing rabbits with S-nitroso-acetylated-cysteine (NO-ac-Cys) conjugates. Using the enzyme-linked immunosorbent assay method, Ab avidity and specificity were determined by competition experiments between NO-ac-Cys-conjugated compounds and other nitrosylated or non-nitrosylated compounds. The resulting cross-reactivity ratios showed that conjugated NO-ac-Cys-BSA was the best recognized compound. These Ab were used for an in vitro study of the kinetics of NO-derived compounds from activated murine macrophages. Anti-NO-ac-Cys Ab inhibited the antimicrobial effect of activated macrophages on the extracellular parasite, T. musculi. Moreover, the L-arginine-dependent antiparasitic activity of supernatants from Calmette-Guerin bacillus-activated macrophages required the presence of albumin and was also inhibited by anti-NO-ac-Cys Ab, showing the effector role of S-nitroso-albumin.

Monoclonal anti-idiotypic antibodies as probes for common idiotopes shared by anti-"benzo(a)pyrene-like" IgA of cancer patients and rabbit anti-conjugated benzo(a)pyrene antibodies.

Anti-"benzo(a)pyrene [B(a)P]-like" IgA [referred as idiotypic antibodies (Abl)] from cancer patients' sera were found to react with conjugated B(a)P and a monoclonal anti-anti-conjugated B(a)P, internal image of conjugated B(a)P called AIB1 (referred to as Ab2 beta). These IgA were used to raise mouse monoclonal anti-idiotypic antibodies (Ab2). A monoclonal Ab2 called AIK1 was characterized as the internal image of a "B(a)P-like" structure. As shown by competitive experiments, AIK1 inhibited the reaction between Ab1 from a rabbit anti-conjugated B(a)P serum- and its relevant internal image, AIB1. Furthermore, AIB1 inhibited the reaction between AIK1 and anti-"B(a)P-like" IgA from cancer patients' sera. These observations confirmed the cross-reactivity between idiotypic determinants of human anti-"B(a)P-like" IgA and rabbit anti-conjugated B(a)P antibodies (Ab). This result was reinforced by a correlation between the anti-"B(a)P-like" IgA levels found in cancer patients' sera using indirect ELISA method with conjugated B(a)P, AIB1 and AIK1 coated on well-plates.

Indirect evidence for nitric oxide involvement in multiple sclerosis by characterization of circulating antibodies directed against conjugated S-nitrosocysteine.

Converging data suggest that nitric oxide (NO) production by cytokine-induced immune cells in demyelinating lesions is involved in multiple sclerosis (MS). High levels of NO may complex to suitable amino acids, causing an immune response against the formed neo-epitopes. By testing MS sera with chemically defined nitroso-amino acids conjugated to carrier protein in ELISA, we observed a significant antibody reaction against the S-nitroso-cysteine epitope. The MS antibody response was exclusively of IgM isotype with an avidity of 8 x 10(-7) M. Sera of all clinical MS forms showed a significantly elevated antibody titer versus sera from healthy subjects or from patients affected with other neurological and autoimmune diseases. The detection of circulating antibodies to a conjugated S-nitroso-cysteine epitope provides indirect evidence for NO involvement in MS.

Identification of a benzo[a]pyrene-like binding protein involved in circulating immune complexes of patients with mammary tumors.

Anti-benzo[a]pyrene (B[a]P)-like autoantibodies (autoAb) have been characterized in sera of patients with epithelial tumors. Circulating immune complexes (CIC) from these sera have been analysed after polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions. Immunoblotting was performed using a monoclonal antiidiotypic antibody (Ab), internal image of conjugated B[a]P called AIB1 and anti-human immunoglobulins (Ig). An immunoreactivity was seen only with AIB1 Ab, suggesting the presence of a 'B[a]P-like' binding protein. Additional studies showed that this immunoreactivity is not associated with an 18- to 20-kDa protein previously identified in the same CIC.

Circadian change of VIP mRNA in the rat suprachiasmatic nucleus following p-chlorophenylalanine (PCPA) treatment in constant darkness.

Neuronal activity of the suprachiasmatic nucleus (SCN) is known to be regulated by two major extrinsic factors conveyed by three anatomically distinct pathways to the SCN: photic stimulus by the direct retinohypothalamic tract (RHT) and the indirect geniculohypothalamic tract (GHT), and information from the brainstem by ascending forebrain serotonergic (5-hydroxytryptamine: 5-HT) tract. It has been shown that VIP mRNA level in neurons of the SCN is altered by external light, but remains stable in constant darkness. In the present study, by using the in situ hybridization technique combined with computer-assisted image analysis, we examined VIP mRNA expression in the SCN of rats in which the two major factors were eliminated, i.e. photic stimulus by exposing animals in total darkness and 5-HT transmission by three-day successive administration of p-chlorophenyl-alanine methylester (an inhibitor of tryptophan hydroxylase, 200 mg/kg, daily). In saline-treated controls, VIP mRNA levels remained almost constant throughout the day. In contrast, in PCPA-treated rats, a significant rhythm of VIP mRNA was observed with a peak at CT 4 and a trough at CT 20. These observations suggest that the removal of photic and 5-HT influence induces VIP mRNA rhythm in the SCN, indicating that VIP mRNA is controlled not only by photic information but also by the circadian clock.

Molecular detection of methionine in rat brain using specific antibodies.

In order to study the localization of methionine in rat brain, an immunological approach was developed by raising antibodies directed against this amino acid. Methionine was conjugated to bovine serum albumin (BSA) or human serum albumin (HSA) via glutaraldehyde. The conjugates were then reduced by sodium borohydride and injected alternately into rabbits. Antibody affinity and specificity were evaluated using an adapted ELISA method, by competition experiments between conjugated methionine and related conjugated compounds, pre-incubated with anti-methionine antibodies diluted at 1/20,000. The resulting cross-reactivity ratios, calculated at half-displacement, showed that glutaraldehyde-methionine conjugate (methionine-G-BSA) was the best recognized compound. Non-reduced methionine conjugate (methionine=G=BSA) and the related-conjugated molecules such as homocysteine, homocysteic acid, cysteine, cystathionine and glutamate were not recognized at all. Antibodies to methionine were directed against a glutaraldehyde-methionine epitope and their very high affinity and specificity made them reliable tools for molecular detection of methionine in rat brain. Using purified antibodies diluted at 1/20,000, motoneurons were found to be the most methionine-immunoreactive cell bodies in glutaraldehyde-fixed rat brain sections.

Chemically induced sarcomas in Sprague-Dawley rats: dose effects on autoantibody levels and tumor progression.

We previously reported that a single injection of 2 mg benzo[a]pyrene (B[a]P) induced seric anti-phosphatidylinositol (PtdIns) autoantibodies (autoAb) and highly malignant sarcomas with 100% efficiency in Sprague-Dawley (SD) female rats. To evaluate the effects of lower doses, we administered a single dose (from 0.125 to 1 mg) of B[a]P into such rats. In all cases, we noted significantly high levels of anti-PtdIns autoAb, with clinically palpable tumors appearing around Day 100. Tumors grew more slowly than they did in the previous model (2 mg B[a]P). Both anti-PtdIns autoAb levels and first appearance of the tumor seemed to be independent of carcinogen doses. Conversely, tumor evolution seemed to depend on B[a]P doses.

Antibodies to reduced glutathione.

Reduced glutathione was conjugated to carrier proteins with glutaraldehyde. Conjugates were reduced by sodium borohydride and injected into rabbits. Using an enzyme-linked immunosorbent assay, antibody affinity and specificity were determined by competition experiments between glutathione conjugate and related conjugated compounds. The resulting cross-reactivity ratios, calculated at half-displacement, showed that conjugated glutathione was the best recognized compound. Non-reduced glutathione conjugate was 50 x less recognized. The other related conjugates were not recognized at all. Thus, the high affinity and relative specificity make these antibodies potentially valuable tools for immunohistochemical detection of reduced glutathione in glutaraldehyde-fixed rat brain. Using purified antisera diluted at 1/5000, reduced glutathione was preferentially visualized in nerve fibers of cortex, cerebellum and spinal cord. These results suggest that concentration of GSH in rat CNS are higher in nerve fibers than in neuronal perikaryons.

Curative effects on rat sarcomas obtained after a treatment combining two monoclonal antibodies.

The effects of a treatment involving two monoclonal antibodies (Ab) were evaluated on benzo(a)pyrene [B(a)P]-induced malignant sarcomas in Sprague-Dawley female rats. These Ab were, respectively, an anti-anti-conjugated B(a)P AB, an internal image of conjugated B(a)P, called AIB1, and an anti-conjugated L-DOPA Ab. They were biweekly injected into animals with small clinically palpable tumors. Anti-'phosphatidylinositol-like' autoantibody (autoAb) levels which were significantly higher in B(a)P-treated rat sera were decreased after Ab treatment. Tumor growth was slowed down compared with that of controls and animal survival was increased. This treatment was more efficient than that involving AIB1 alone. The anti-conjugated L-DOPA Ab may play a role in neovascularization, which is known to be critical for tumor growth.

Neuritic GABAergic synapses in insect neurosecretory cells.

The localization of synaptic gamma-aminobutyric acid (GABA) receptors on cockroach dorsal unpaired median (DUM) neurons of the last abdominal ganglion was investigated. These neurosecretory cells, mainly octopaminergic, possess a soma located on the dorsal midline of the ganglion, from which emerges a short primary neurite dividing into two symmetrical lateral branches on both lateral edges of the ganglion. GABA pressure ejections onto the soma and onto the neuritic arborization elicited hyperpolarizations. Moreover, electrical stimulation of the anterior connectives evoked a postsynaptic potential, mainly inhibitory. This response and the GABA hyperpolarization of the neuritic field are antagonized by lateral application of picrotoxin while soma GABA hyperpolarization remained unchanged. This suggests that there are two kinds of GABA receptors located (i) onto the soma membrane of the DUM neurons and called extrasynaptic receptors, (ii) on the neuritic arborization, called synaptic receptors and implicated in the connection between neurons coming from the anterior part of the nervous system and the DUM cells. Immunohistological double staining technique reinforced the electrophysiological results by showing the presence of GABA-like immunoreactive processes next to octopamine-like immunoreactive ones.

[Effects of monoclonal anti-idiotypic antibody, internal image of benzo(a)pyrene on sarcoma in rats]. / Effets d'un anticorps monoclonal anti-idiotypique, image interne du benzo(a)pyrène sur des sarcomes de rats.

The effects of a monoclonal anti-idiotypic antibody (Ab), internal image of conjugated benzo(a)pyrene [B(a)P], called AIB1, on B(a)P-induced malignant sarcomas in Sprague-Dawley (SD) female rats were evaluated at different times after B(a)P-administration. As previously described, circulating anti-"phosphatidylinositol (PtdIns)-like" autoantibody levels were found to be significantly higher in B(a)P-treated rat sera. They were decreased after AIB1 Ab-treatment. Furthermore, the tumor growth was slowed down compared with that of controls and animal survival was slightly increased. These results showed the relationships between metabolic pathways involving possible "B(a)P-like" endogenous ligand/cytosolic receptor(s) and the PtdIns.

A monoclonal anti-idiotypic antibody with an internal image of a "phosphatidylinositol-like" structure derived from anti-"phosphatidylinositol-like" IgG from the sera of patients with proliferative malignancies.

Using an enzyme-linked immunosorbent assay (ELISA) with phosphatidylinositol (PtdIns) coated on well-plates, high levels of anti-"PtdIns-like" autoantibodies (autoAbs) have been previously described in sera of cancer patients. These anti-"PtdIns-like" autoAbs were purified and injected into BALB/c mice. A monoclonal anti-idiotypic antibody (Ab2), internal image of human endogenous "PtdIns-like" structure called AIPI, was selected. Then the immunological binding in sera of cancer patients was evaluated on AIPI coated on well-plates. Using this indirect ELISA method, we found a statistically highly significant immunological binding in sera of patients with epithelial tumors, which correlated with that previously found with the PtdIns molecule coated on well-plates. Moreover, AIPI mimics an endogenous structure closely associated with PtdIns specifically encountered in epithelial proliferative disease.

Curative effects of all-trans-retinoic acid on rat sarcomas.

The effects of all-trans-retinoic acid (all-trans-RA) on benzo(a)pyrene [B(a)P]-induced malignant sarcomas in Sprague-Dawley female rats were evaluated. Ninety-eight days after B(a)P administration, all-trans-RA was daily injected to animals with or without clinically palpable tumors. The growth of tumors was slowed down compared with controls. Magnetic resonance imaging analyses showed that all-trans-RA-treated rat tumors presented early necrotic areas. Animal survival was slightly increased. Anti-phosphatidylinositol autoantibody levels which were significantly higher in B(a)P-treated rat sera were not modified by all-trans-RA treatment.